The objective of this project is to study the influence of neurons on astroglial differentiation. To analyze the contribution of neuronal interactions to glial morphology and proliferation, we will purify granule neurons and astroglia harvested from early postnatal mouse cerebellum into highly enriched cell fractions and recombine them in varying ratios. We will then correlate the number of neurons and occurrence of neuron-glial contacts with astroglial morphology by quantitative morphometric analysis of immunostained cells. The proliferation rate of the astroglia will be measured by a cytofluorograf modular flow cytometer system. Time-lapse video microscopy will be used to analyze whether individual astroglial cells, cultured alone or in the presence of varying numbers of neurons, change their shape or have stable, identifiable morphological types over time in culture. To begin to analyze the mechanism of neuronal regulation of astroglial differentiation, we will test for trophic factors in the medium, use a fluorescence assay for gap junctions and analyze whether the intracellular cAMP or Ca++ levels of the astroglia change when neurons are present. To distinguish the contribution of the astroglial genome versus that of neuronal interactions, we will recombine the enriched granule neuron fraction with purified weaver mouse astroglia. To analyze the influence of normal neurons on the proliferation rate of transformed astroglia, we will add normal neurons to clones of rodent C6, G26 and human U251 glioma cell lines and quantitate the proliferation rate of the glioma cells.